Seasonal Fluctuations in Iron Cycling in Thawing Permafrost Peatlands

In permafrost peatlands, up to 20% of total organic carbon (OC) is bound to reactive iron (Fe) minerals in the active layer overlying intact permafrost, potentially protecting OC from microbial degradation and transformation into greenhouse gases (GHG) such as CO2 and CH4. During the summer, shifts in runoff and soil moisture influence redox conditions and therefore the balance of Fe oxidation and reduction. Whether reactive iron minerals could act as a stable sink for carbon or whether they are continuously dissolved and reprecipitated during redox shifts remains unknown. We deployed bags of synthetic ferrihydrite (FH)-coated sand in the active layer along a permafrost thaw gradient in Stordalen mire (Abisko, Sweden) over the summer (June to September) to capture changes in redox conditions and quantify the formation and dissolution of reactive Fe(III) (oxyhydr)oxides. We found that the bags accumulated Fe(III) under constant oxic conditions in areas overlying intact permafrost over the full summer season. In contrast, in fully thawed areas, conditions were continuously anoxic, and by late summer, 50.4 ± 12.8% of the original Fe(III) (oxyhydr)oxides were lost via dissolution. Periodic redox shifts (from 0 to +300 mV) were observed over the summer season in the partially thawed areas. This resulted in the dissolution and loss of 47.2 ± 20.3% of initial Fe(III) (oxyhydr)oxides when conditions are wetter and more reduced, and new formation of Fe(III) minerals (33.7 ± 8.6% gain in comparison to initial Fe) in the late summer under more dry and oxic conditions, which also led to the sequestration of Fe-bound organic carbon. Our data suggest that there is seasonal turnover of iron minerals in partially thawed permafrost peatlands, but that a fraction of the Fe pool remains stable even under continuously anoxic conditions.


Synthesis of Ferrihydrite-coated Sand.
Quartz sand with a grain size of 0.4-0.8 mm (Carl Roth GmbH + Co.KG, Germany) was used. The sand was pre-treated by first autoclaving the sand at 121°C for 20 minutes, followed by washing with 1 M HCl (24 h, completely covered) and finally with Aqua Regia for 5 min to further improve Fe coating efficiency by increasing the surface area, as has been done previously 1, 2 . Afterwards the sand was washed with MilliQ water and dried at 60°C.
The Fe(III) oxyhydroxide 2-line ferrihydrite (FH) was synthesized in the presence of the sand by precipitation from a Fe(NO3)3*9H2O solution by adding 1 M KOH 1 . The Fe(NO3)3*9H2O solution was added to 500 g of sand and stirred manually by hand as KOH was added dropwise until a pH of 7.3 was reached. The mixture was then left without stirring. After two hours, the pH was readjusted to 7.5 and the mixture left overnight on a rolling shaker (15 rpm), as has been done previously 1 . Finally, the mixture was washed with MilliQ water and dried at 40°C to avoid temperature-induced modifications of the precipitates 1 .
In the end, the fresh synthesized ferrihydrite-coated sand yielded an iron content of 2.19±0.26 mg poorly crystalline Fe(III) per g sand, determined by 0.5 M HCl extraction, performed in triplicates, followed by Fe quantification in the extract by Ferrozine assay 3 . Treating the unexposed sand in the same manner as the sand incubated within the soils (i.e. transport to the field and back, and stored at room temperature), formed a more crystalline Fe(III) phase (1.01±0.14 mg Fe(III) per g sand), which was only extractable by 6 M HCl, not extractable with 1 M Na-acetate and 0.5 M HCl. As also previously stated 1 , the FH coating increased the specific surface area (SSA) from 0.07 m 2 per g of the initial pure sand to 1.49 m 2 per g of the FH-coated sand (21 times higher than for its uncoated precursor).
Ferrihydrite Bags. The FH-coated sand was packed in Teflon bags (polytetrafluoroethylene (PTFE); or Teflon) with 0.1 mm thickness and manually pierced with needles of 0.55 mm diameter under sterile conditions in a Biological Safety Cabinet (BSC). The bag was closed with a cable tie at the top and with a long plastic line, which was later used to mark the position of the bag at the surface and pull it out after incubation. The Teflon along with additional equipment (FH coated sand, cable ties) was autoclaved (121°C, 1 bar pressure, 20 mins) prior to use, brought into the BSC cabinet and exposed to UV-light for 15 minutes. The Teflon was chosen based on the following suitable properties: heat (up to 250°C) and cold (until -196°C) resistant; unaffected by most chemicals, especially iron; no adhesive forces and weather.
The bags filled with FH-coated sand were stored at room temperature in sterile plastic bags for 3-4 weeks before being transported to the field under sterile conditions. Sequential Fe Extraction. 0.5 g of homogenized sand from each thaw stage and collection periods was added under anoxic conditions (100% N2, remaining O2 <100 ppm) into Eppendorf tubes. As previously described 6 , samples were centrifuged (5 min, 12,000 g) and the porewater (supernatant) was removed. 1 mL of anoxic 1 M Na-acetate solution was added to the pellet, mixed and incubated for 24 h in the dark. Then, the sample was centrifuged (5 min, 12,000 g) and the supernatant was collected and stored anoxically in the dark at 4°C until further analysis.
To the pellet, 1 mL of anoxic 0.5 M HCl was added, mixed and incubated in the dark under anoxic conditions for 2 h. After centrifugation again, the supernatant was removed and stored anoxically at 4°C until further analysis. To the remaining pellet, 1 mL of anoxic 6 M HCl was added, mixed and incubated for 24 h in the dark as the last extraction step 6  Primers were trimmed, and untrimmed sequences were discarded (< 13%, on average 9.6%) with Cutadapt v2.6 13 . Adapter and primer-free sequences were imported into QIIME2 version 2019.10.0 14 , processed with DADA2 version 1.10.0 15 to eliminate PhiX contamination, trim reads (before the median quality drops below 38, i.e. position 137 in forward reads and 194 in reverse reads), correct errors, merge read pairs, and remove PCR chimeras; ultimately, in total 937 amplicon sequencing variants (ASVs) were obtained across all samples. Alpha rarefaction curves were produced with the QIIME2 diversity alpha-rarefaction plugin, which indicated that the richness of the samples had been fully observed. A Naive Bayes classifier was fitted with 16S rRNA (gene) sequences extracted with the PCR primer sequences from the QIIME compatible, 99%-identity clustered SILVA v132 database 16 . ASVs were classified by taxon using the fitted classifier 17 . 45 ASVs that classified as chloroplasts or mitochondria were removed, totalling to < 0.5% (average 0.36%) relative abundance per sample, and the remaining 892 ASVs had their abundances extracted by feature-table (https://github.com/qiime2/q2feature-table).
DNA extraction was only successful for the bags deployed in the fully thawed fen until late summer.
Isolation of Fe(III)-reducing bacteria was performed with anoxic media and supplies (5 mM acetate and 5 mM lactate) via multiple rounds of extinction, as previously described 18 . The headspace in the dilution series was N2:CO2 (90:10). To the first tube of a dilution series, 1g of FH-coated sand was added, and a 10x dilution series up to a dilution of 10 -12 was prepared. To     In early summer, the semi-wet bog and waterlogged fen areas were completely water-saturated.
During summer, bog areas became drier due to increasing drainage caused by active layer deepening and decreasing volumetric soil water content in the upper 10 cm. in bog in blue and in fen in green. The first 1-2 days represent the equilibrium phase after installing the redox probes in the field. Values above +300 mV are considered as oxic conditions. Values of +300 mV to +100 mV are considered as weakly reduced and values of +100 to -100 mV as moderately reducing conditions 20 . Towards August, the redox potential in bog increases from 0 mV to above +300 mV which marks a potential shift from Fe(III)-reducing to Fe(II)-oxidizing conditions. Unfortunately, the redox probes disconnected from the battery in mid-August and remote data collection ceased.         Table S1. Organic carbon (OC) to iron (Fe) ratios in the active layer of the partially thawed bog and in the fully thawed fen collected after incubation of 2 months until late summer.